Lipoproteins present in blood are roughly classified into four classes: high-density lipoprotein (hereinafter abbreviated as HDL), low-density lipoprotein (hereinafter abbreviated as LDL), very low-density lipoprotein (hereinafter abbreviated as VLDL) and chylomicron (hereinafter abbreviated as CM) according to their specific gravity. Each class of lipoprotein has a different lipid composition and also has a considerably different function in vivo according to the kind of apoproteins constituting each lipoprotein. Also there exists, as a lipoprotein formed in the process of metabolism from VLDL to LDL, intermediate-density lipoprotein (hereinafter abbreviated as IDL) having a density between those of VLDL and LDL, which is classified as LDL in a broad sense.
In clinical tests, total cholesterol, total triglyceride, HDL cholesterol, LDL cholesterol, apolipoprotein AI, apolipoprotein B, etc. are used as a screening marker for diagnosis of arteriosclerosis. In particular, the frequency of measurement of cholesterol in LDL, which is said to be strongly associated with the formation of arteriosclerosis, has been increasing. On the other hand, there observed many patients having coronary arteriosclerotic lesion without showing a high LDL cholesterol level. In such cases, increase of small and high-density LDL (this LDL is called sdLDL) is observed, and there is a report that sdLDL-C is more closely associated with coronary diseases than LDL-C [see Arteriosclerosis Thrombosis, and Vascular Biology, Vol. 24, p. 558-563 (2004)].
Known examples of methods for measuring sdLDL include the electrophoresis method, the ultracentrifugation method and the fractionation method. The electrophoresis method is a method which comprises carrying out electrophoresis and staining using 2 to 16% non-denatured gradient gel to measure the particle size of LDL, but it involves complicated operations and is unsuitable for wide use. Further, this method is capable of measuring the particle size of LDL but is incapable of determining sdLDL. Also known is a method in which sdLDL is suspended or dissolved according to the difference in ionic strength and sdLDL is measured based on the difference in absorbance (see patent document 1). However, being based on the measurement of turbidity, this method is insufficient in specificity and accuracy, and further, it is capable of measuring the amount of sdLDL but is incapable of measuring sdLDL-C.
Known examples of methods for determining sdLDL-C include the ultracentrifugation method and the fractionation method. The ultracentrifugation method is a method which comprises separating a specific gravity region corresponding to sdLDL in a sample using an ultracentrifuge and measuring cholesterol in the specific gravity region, and it can be a standard method for determination of sdLDL-C. However, this method requires expensive equipments and also requires a very long measurement time and operation skill.
The fractionation method is a method which comprises a first step in which lipoproteins other than HDL and sdLDL in a sample are aggregated using a combination of polyanion and a divalent cation or a monovalent cation, or polyethylene glycol, and the aggregated lipoproteins are removed by centrifugation or filtration using a filter to separate HDL and sdLDL, and a second step in which sdLDL-C in the sample containing the separated HDL and sdLDL is determined (see patent document 2). Although this method can be performed with simplified operations compared with the ultracentrifugation method, it requires a long measurement time because of the necessity of a fractionation operation.
There are many reports on the methods for measuring cholesterol in a specific lipoprotein to be measured by allowing a cholesterol ester hydrolase and cholesterol oxidase or cholesterol dehydrogenase to act specifically on one or plural specific lipoproteins selected from the group consisting of HDL, LDL, VLDL and CM in the presence of surfactants. Recently, there has been reported a method for determination of sdLDL-C which comprises adding enzymes for measurement of cholesterol to a sample in the presence of a polyoxyethylene-polyoxypropylene copolymer or its derivative, allowing the polyoxyethylene-polyoxypropylene copolymer or its derivative to act selectively on sdLDL, and measuring the amount of formed cholesterol (patent document 3).    Patent document 1: Japanese Laid-Open Patent Publication No. 2003-28882    Patent document 2: WO2004/053500 pamphlet    Patent document 3: WO2007/026829 pamphlet